Refined annotation and assembly of the Tetrahymena thermophila genome sequence through EST analysis, comparative genomic hybridization, and targeted gap closure
1 J. Craig Venter Institute (formerly The Institute for Genomic Research), 9704 Medical Center Dr., Rockville, MD, USA
2 Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, USA
3 The Broad Institute, 7 Cambridge Center, Cambridge MA, USA
4 Dept. of Microbiology and Immunology, Cornell University Veterinary Medical Center, Ithaca, NY, USA
5 Joint Sciences Dept., Claremont Colleges, Claremont, CA, USA
6 Dept. of Biomedical Sciences, Missouri State Univ., Springfield MO, USA
7 Molecular and Cell Biology Dept., University of California Berkeley, Berkeley CA, USA
8 Pathology Dept., University of Michigan Medical School, Ann Arbor MI, USA
9 Dept. of Biology, York University, Toronto ON, Canada
10 Dept. of Molecular, Cellular, and Developmental Biology, University of California Santa Barbara, Santa Barbara CA, USA
11 UC Davis Genome Center, Section of Evolution and Ecology, University of California, Davis, CA, USA
BMC Genomics 2008, 9:562 doi:10.1186/1471-2164-9-562Published: 26 November 2008
Tetrahymena thermophila, a widely studied model for cellular and molecular biology, is a binucleated single-celled organism with a germline micronucleus (MIC) and somatic macronucleus (MAC). The recent draft MAC genome assembly revealed low sequence repetitiveness, a result of the epigenetic removal of invasive DNA elements found only in the MIC genome. Such low repetitiveness makes complete closure of the MAC genome a feasible goal, which to achieve would require standard closure methods as well as removal of minor MIC contamination of the MAC genome assembly. Highly accurate preliminary annotation of Tetrahymena's coding potential was hindered by the lack of both comparative genomic sequence information from close relatives and significant amounts of cDNA evidence, thus limiting the value of the genomic information and also leaving unanswered certain questions, such as the frequency of alternative splicing.
We addressed the problem of MIC contamination using comparative genomic hybridization with purified MIC and MAC DNA probes against a whole genome oligonucleotide microarray, allowing the identification of 763 genome scaffolds likely to contain MIC-limited DNA sequences. We also employed standard genome closure methods to essentially finish over 60% of the MAC genome. For the improvement of annotation, we have sequenced and analyzed over 60,000 verified EST reads from a variety of cellular growth and development conditions. Using this EST evidence, a combination of automated and manual reannotation efforts led to updates that affect 16% of the current protein-coding gene models. By comparing EST abundance, many genes showing apparent differential expression between these conditions were identified. Rare instances of alternative splicing and uses of the non-standard amino acid selenocysteine were also identified.
We report here significant progress in genome closure and reannotation of Tetrahymena thermophila. Our experience to date suggests that complete closure of the MAC genome is attainable. Using the new EST evidence, automated and manual curation has resulted in substantial improvements to the over 24,000 gene models, which will be valuable to researchers studying this model organism as well as for comparative genomics purposes.